The overall goal of this procedure is to prepare keratin hydrolysate from chicken feathers by alkaline enzymatic hydrolysis, and to test whether adding of keratin hydrolysate into a cosmetics ointment base improves skin barrier function. This method can help answer questions in the slaughterhouse keratin byproducts field such as utilization of chicken poultry feathers for preparation of keratin hydrolysate, which can be used in cosmetic formulations. The main advantage of this technique is that keratin hydrolysate is a high-quality and acts as a very good occlusive and humectant in cosmetic formulations.
Demonstrating the procedure of applying tested formulations at each spot of the four arm sides will be Jana Pavlackova, a co-author of this publication. To begin, grind 50 grams of dried feathers in a cutting mill into a final fineness of 1.0 millimeters. Next, degrease the feathers in a stainless steel 27-liter boiler container with temperature control.
Mix the feathers with water preheated up to 40 plus or minus two degrees celsius in a weight ratio of one to 75. Add a lipolytic enzyme in a dose of 1.5 to 2.0%and gently stir the contents with an overhead stirrer for five minutes. Adjust the mixture pH to 9.0 plus or minus 0.2 by adding 1%sodium hydroxide or 1%phosphoric acid solution.
This is the pH value corresponding to the maximum activity of the lipolytic enzyme. After stirring the mixture for five minutes with an overhead stirrer, check the pH level using a laboratory bench pH/mV meter and readjust the pH level. Then, gently stir the mixture with an overhead stirrer for 24 hours at 40 plus or minus 0.5 degrees celsius.
Filter the mixture through a fine sieve and wash the degreased feathers with a stream of fresh running cold water. Then, dry the feathers on a flat plate at 50 degrees celsius in a drying chamber overnight. The next day, perform the first stage of the chicken feather hydrolysis.
Mix the feathers with 0.3%potassium hydroxide water solution in a weight ratio of one to 50, and gently stir with an overhead stirrer at 60 plus or minus 0.5 degrees celsius for 24 hours. After finishing the first stage of hydrolysis, adjust the pH of the mixture to the level corresponding to the maximum activity of the proteolytic enzyme with 10%phosphoric acid, which in this case, is a level of 9.0 plus or minus 0.2. Perform the second stage of chicken feather hydrolysis.
Add the proteolytic enzyme to the mixture in a dose of 5.0%Gently stir the mixture with an overhead stirrer at 60 plus or minus 0.5 degrees celsius for eight hours. Then, heat the mixture in the same stainless steel 27-liter boiler container to the boiling point and boil for 10 minutes to inactivate the enzyme. Separate the solution of KH from the undissolved remnant by filtering it through low-density filter paper on a Buchner funnel with slight vacuum pressure.
Pour 400 milliliters of the KH solution into 12K molecular weight cutoff dialysis tubing in order to remove small peptides and salts. Dialyze the solutions against four liters of distilled water in a plastic bucket for 80 hours at room temperature. Change the distilled water after 18, 36, and 60 hours.
Cast the dialyzed solution of KH on an antiadhesive plate at a ratio of 500 milliliters to a 1, 000-square-centimeter plate area. Vacuum dry the solution to a thin film at 40 plus or minus 0.5 degrees celsius overnight. After scraping the dried film, grind it to form a fine powder and keep it in a closed vessel in a desiccator.
Next, prepare cosmetic formulations containing two, four, and 6%KH in accordance with the base weight of the ointment. To do so, weigh the amount of KH powder into a polyethylene vessel. Then, add the ointment base at an amount that ensures the total weight of the formulation equals 50 grams.
Homogenize the mixture with a three-bladed laboratory blender for 10 minutes at 134.16 g before mixing using a mechanical overhead stirrer. Maintain the prepared formulations at five plus or minus one degree celsius, and warm them at room temperature for two hours prior to use. Place five strips of filter paper into a physiological solution of 0.90%sodium chloride and leave them for approximately one minute in the solution.
Apply two strips to the inner side of the right forearm, and three to the inner side of the left forearm, and fix them for four hours with adhesive plasters. After four hours, remove the strips and demark the areas with a permanent pen. Prepare to apply 0.1 milliliters of the tested formulations at each spot of the degreased forearm sites.
On the left forearm, do not add anything to the first site as it is the control. Then, apply the ointment base only to the second site. Use syringes and spread it over the entire marked surface.
Similarly, spread the ointment base plus 2%KH onto the third site. Finally, apply the ointment base plus 4%KH and ointment base plus 6%KH to the right arm. Measure each sample at each site and each interval.
Take five readings with the skin hydration meter probe for skin hydration, 15 readings with the tool meter probe for skin transepidermal water loss, and one reading with the skin pH meter probe for skin pH. Process the resulting readings as described in the text protocol. Shown here is a representative photograph of keratin hydrolysate showing its yellow color.
Keratin hydrolysate is easily soluble in water, has high protein content, and fulfills requirements for cosmetic-grade hydrolysates. Electrophoretic profiles of low molecular protein standard, high molecular protein standard, and keratin hydrolysate, show that keratin hydrolysate has wide molecular weight heterogeneity. Once mastered, this technique can be done in 150 hours if it is done properly.
After watching this video you should have a good understanding of how to prepare keratin hydrolysate and how to apply it in cosmetic formulations. After its development, this technique paved the way for researchers in the field of protein byproduct treatment to explore utilization of chicken feathers in cosmetic industry.
